FIGURE 8.

Subcellular localization of DGKη-PH in COS-7 cells treated with hyperosmotic shock in the presence of synaptojanin. A, AcGFP-tagged DGKη-PH was co-expressed with FLAG-tagged synaptojanin in COS-7 cells. After 48 h of transfection, the cells were incubated with 500 mm sorbitol in DMEM for 30 min. The cells were fixed with 3.7% formaldehyde, stained with anti-FLAG and Alexa Fluor 594-conjugated secondary antibodies, and observed using confocal laser scanning microscopy. Scale bars, 20 μm. B, the percentages of cells exhibiting translocation of AcGFP-tagged DGKη-PH to the plasma membrane in the presence or absence of synaptojanin were scored. More than 30 cells expressing AcGFP-tagged DGKη-PH were counted in each experiment. The results are the means ± S.D. of three independent experiments. The statistical significance (versus −synaptojanin +sorbitol) was determined using Student's t test (***, p < 0.005). Error bars represent S.D.